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1. Field of the Invention
The present disclosure relates generally to the field of DNA amplification and more particularly to the field of amplifying any stretch of DNA in a sequence-independent manner.
2. Description of Related Art
The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
It is well known that there is often an association between genetic variation and phenotype manifestation. Genetic variations and their associated phenotypes are studied using various methods of genotyping genomic DNA. A Single Nucleotide Polymorphism (SNP) is a single nucleotide variation at a specific location in the genome of different individuals. SNPs are stable genetic variations frequently found in genes, and contribute to the wide range of phenotypic variations found in organisms. SNP genotyping is useful in developing detailed genetic and physical maps of chromosomes. Genotyping densely distributed SNP markers across the different chromosomes of an individual can help reveal statistically significant correlations between chromosomal loci and phenotypic expression. Extensive genotyping, however, requires not only a simple and rapid way for obtaining, shipping, storing, and sorting large amounts of genetic material, but also convenient and high-throughput methods for extracting large quantities of DNA from these samples.
There are a variety of available methods for obtaining and storing tissue and/or blood samples. These alternatives allow tissue and blood samples to be stored and transported in a form suitable for the recovery of genomic DNA from the samples for genotype analysis. DNA samples can be collected and stored on a variety of solid mediums, including Whatmann(copyright) paper, Guthrie cards, tubes, swabs, filter paper, slides, or other containers. When whole blood is collected on filter paper, for example, it can be dried and stored at room temperature.
One known and more frequently used method for securing and storing DNA is described in U.S. Pat. No. 5,496,562. This method involves storing dried animal blood samples on chemically treated filter paper, called FTA paper, that protects genomic DNA from degrading (commercially available as FTA(trademark) paper by Whatman(copyright)). FTA paper is light weight and easy to store, which makes it a popular choice for collecting genetic material and samples. Samples on FTA paper are conveniently stored and shipped at room temperature.
All of the materials available to those of skill in the art for storing blood or other tissues containing DNA have limitations. For example, the amount of tissue or blood collected may be very limited, which makes wide-scale and high-throughput genotyping impractical and expensive. For example, despite the widespread use of FTA paper, its usefulness is limited because the stored bloodstains contain only a small amount of genomic DNA. A 6.0 cm2 piece of FTA paper only preserves approximately 100 xcexcl of blood, equivalent to approximately 1.0 xcexcg of DNA. While it is possible to extract genomic DNA from a larger piece of FTA paper, the size of the paper makes it cumbersome to manipulate in the small wells of a 96-well plate or a 384-well plate, both of which are important tools for high-throughput screening of large numbers of DNA samples. Therefore, the usefulness of a tool like FTA paper has been restricted to low-volume genotyping.
The limited amount of DNA stored on FTA paper also makes it impractical for genotyping multiple polymorphisms and genetic loci in a single organism. The FTA paper sample can be cut into smaller pieces for genotyping multiple SNPs; a small circle of 1.0-2.0 mm2 diameter of the sample contains about 1-5 ng of genomic DNA, which is sufficient for one polymerase chain reaction (PCR). But this approach is undesirable because it requires repetitive cutting, sorting, and extracting of the FTA paper, which is not only tedious but also prone to human error. For a genomic scan of hundreds or thousands of SNPs, the task of cutting and analyzing DNA samples stored on FTA paper is an insurmountable barrier for researchers.
Additionally, the strong adherence between DNA and FTA paper makes DNA extraction for analysis difficult. Although proteinase K and endonuclease digestion can facilitate DNA release as suggested by the manufacturer, this approach is too complicated and expensive for high-throughput operations. The commercially available FTA Purification Reagent, which can be used to prepare DNA stored on FTA paper for analysis by PCR(trademark) yields inconsistent results. For example, often no specific DNA amplification is achieved after the DNA sample is processed using this reagent, which is unacceptable in a high-throughput operation. The manufacturer also suggests that the strong adhesion of DNA to FTA paper allows for repeated genotyping of DNA stored on FTA paper. Notwithstanding the fact that PCR efficacy for xe2x80x9crecycledxe2x80x9d FTA paper has not been fully tested, cleaning the tiny FTA papers between consecutive SNP PCRs is impractical for high-throughput processing. The small pieces of floating filter paper are difficult to wash by conventional aspiration, and they tend to clog aspiration needles or pipette tips. Further, small pieces are easily lost during the cleaning process. Finally, repeated pipetting of PCR products has an associated risk of cross contamination among different wells.
The shortcomings associated with small samples of blood or tissue from an organism are overcome by efficient methods of whole genomic DNA amplification. For example, whole genomic DNA amplified from the small amounts of DNA sample stored on FTA paper could be used in multiple PCR reactions to extensively genotype various polymorphisms such as SNPs found in a single organism in a high-throughput screening process. Nevertheless, while several methods for whole genome amplification have been proposed and successfully used for various applications in the past, these methods are generally inefficient, complex, and expensive. Therefore, the need exists for a simple and cost effective way of amplifying genomic DNA from small samples of blood or tissue.
One of the first methods for amplifying DNA was the linker adaptor-mediated PCR (LAM-PCR) approach, which has been applied to microdissected chromosomes (Zhou et al., Bio Techniques 28:766-774, 2000; Albani et al., Plant J 4(5):899-903, November 1993), yeast artificial chromosome (YAC) DNA (Sutcliffe et al., Genomics 13(4):1303-6, 1992), and genomic DNA (Kinzler et al., Nucleic Acids Res 25:17(10):3645-53, May 1989). In this approach, the starting DNA is first digested with a restriction enzyme, usually an enzyme with a four base recognition sequence. After inactivation of the restriction enzyme, a known sequence (either an adaptor or a synthetic linker) is ligated to the ends of the DNA fragments generated by the restriction-enzyme digest, providing primer binding sites for PCR amplification. The DNA can then be amplified by PCR using primers that are complementary to the sequence of the adaptor or linker.
Unfortunately, the usefulness of LAM-PCR is limited because it involves multiple steps, including DNA fragmentation, adaptor or linker ligation, and PCR amplification. These steps make this process both laborious and expensive for high-throughput genotyping. An additional shortcoming of this method is that sequences that do not contain the recognition sequence of the restriction enzyme used at appropriately spaced intervals will not be amplified by PCR because the regions will be too long to amplify. This method is also time-consuming and cumbersome because of the extensive manipulations of DNA necessary to attach the known sequences to both ends of the fragments, especially when applied to small quantities of DNA, such as microdissected chromosomal pieces, or DNA found in bloodstains or small samples of tissue.
Another more restrictive method available for amplifying genomic DNA uses inter-ALU PCR (more generally known as inter-repetitive element PCR), which relies on the presence of appropriately spaced and oriented ALU repetitive elements or other repeated sequences. Inconsistent results are obtained, however, with low complexity DNA sources such as YACs, cosmids, or phage, because of the low incidence of these repeat sequences. Other inconsistencies arise because repeated sequences do not occur uniformly throughout the genome, and thus a sequence of interest occurring in an area in which the necessary repeated sequences are rare or absent will not be amplified. Another major limitation of this method is that it is species specific. For example, the use of this method is restricted to DNA of the species from which the repetitive elements are derived and for which the PCR primers were constructed.
A method called degenerated oligonucleotide-primed PCR (DOP-PCR) utilizes partially degenerated sequence (6 out of 21) and repeated thermocycling Telenius, et al., Genomics 13(3):718-25, 1992. In the DOP-PCR method, the first rounds of PCR amplification have a low primer annealing temperature of around 30xc2x0 C. The primer used consists of a random hexamer that is flanked on the 3xe2x80x2 side by a defined hexamer and on the 5xe2x80x2 side by a defined sequence. Because the 3xe2x80x2 end of the primer has a defined hexamer, the target sequence must match this hexamer in order to amplify. Therefore, the number of sequences that will be amplified by this method are limited. The inadequacy of the DOP-PCR method is further demonstrated when it is applied to DNA sources of limited complexity such as YACs, cosmids, or phage inserts. The resulting product is not a smear on a ethidium bromide stained agarose gel (as occurs with randomly amplified DNA), but rather distinct bands, indicating that hybridization occurs at relatively few sites and thus sequence independent amplification is not achieved.
Another attempt to amplify genomic DNA was a method termed primer-extension preamplification (PEP) (Zhang et al., Proc. Natl. Acad. Sci. USA 89:5847-5851, 1992). The PEP method utilizes 15 base pair (bp) random oligonucleotides and repeated thermocycling to randomly prime multiple sites in the genomic DNA for PCR. A method utilizing 6 base pair (bp) random oligonucleotides and PCR has also been reported (Peng et al., Clin Pathol 47:605-608,1994). Although both PEP and DOP-PCR have been employed in several specific applications, they are consistently hampered by their relatively low amplification efficiency (Wells et al., Nucleic Acids Res 27:1214-1218, 1999). A possible explanation for this low efficiency is that because the primers contain random nucleotides and therefore form a large spectrum of different oligonucleotides, the effective concentration of any specific primer is very low, which may limit the exponential amplification of PCR. Additionally, the non-specific binding of the random oligonucleotides tends to initiate DNA synthesis within the PCR products of previous rounds. Therefore, the size of the PCR products decreases constantly with each additional round of PCR amplification, which renders the final PCR products very small, especially when a large number of PCR cycles are performed. These small PCR products are not as useful as larger pieces of amplified DNA for the subsequent genetic analysis of the DNA or genotype analysis.
Another method of genomic DNA amplification, termed tagged random PCR, was described by Grothues et al. (Nucleic Acids Res 21:1321-1322, 1993) and Wong et al. (Nucleic Acids Res 24:3778-83,1996). This method attempts to overcome the shortcomings associated with PEP and DOP-PCR by separating random priming and PCR amplification into two steps and amplifying whole genomic DNA with a single PCR primer. In the first amplification step, tagged random primers consisting of a random 6 bp or 9 to 15 bp 3xe2x80x2 tail and a constant 17 to 22 bp 5xe2x80x2 head indiscriminately prime the genomic DNA. Next, unincorporated tagged primers are removed by gel filtration using a Biogel P100 spin column or a Centricon-100 spin column. In the second amplification step, the DNA molecules fitted with the 5xe2x80x2 constant head and its reverse complement at both ends are amplified by PCR. Although the scheme can amplify whole genomic DNA, its multiple steps of reaction and purification are too complex and expensive for high-throughput screening.
The construction of libraries from microdissected chromosomal bands is an elegant way to obtain DNA probes from genomic regions of particular interest. The applicability of this approach has been restricted by the time consuming and technically difficult process of amplifying DNA from microdissected material. Traditionally, DNA from 20 to 30 microdissected chromosomal bands is collected in a small droplet. The DNA is then subjected to various manipulations before it is used for PCR amplification. These manipulations include phenol/chloroform extractions, restriction enzyme digestion and ligation to a vector or linker (LAM-PCR). These steps must be performed in very small volumes on the stage of a microscope with specialized equipment (Kao et al., Proc Natl Acad Sci USA 1:88(5):1984-8,1991), which severely limits the usefulness of this technique.
Yeast artificial chromosomes (YACs) are ideal vectors for the detailed mapping of large stretches of DNA. One of the main disadvantages of the YAC cloning system is that there have been no methods available to purify YAC DNA in large quantities. High molecular weight DNA can be prepared from yeast clones carrying YACs and the YACs can be isolated on a pulsed field gel. This approach, however, yields only very small amounts of pure YAC DNA. This is a major disadvantage because many important uses of these large inserts, e.g. screening of cDNA libraries or Fluorescent in situ hybridization (FISH) analysis, require larger amounts of purified YAC DNA.
Thus, a need exists for a more efficient and inexpensive method for amplifying DNA samples that is compatible with high-throughput screening, is sequence independent, applicable to any type of DNA, useful for amplifying DNA from any species, and most important, capable of amplifying extremely limited amounts of DNA. There is also a need for an amplification process that is simple (to avoid the problem of PCR contamination), has high fidelity in reproducing genetic material, and has a low rate of distortion of amplified sequences.
The present disclosure seeks to overcome the drawbacks inherent in other methods of DNA amplification by providing a simple and direct method for amplifying DNA. The method of the present disclosure preferably amplifies DNA in a sequence-independent manner using a single reaction mixture and a single programmable thermocycling reaction. This method can be used to amplify trace amounts of DNA, including genomic DNA from small tissue or blood samples, such as fine needle aspirates or single tissue sections, or even from a single cell. The single reaction mixture used in this method also greatly reduces the risk of sample contamination and facilitates high-throughput screening, and in a preferred embodiment a single heat-stable DNA polymerase is included to amplify all DNA in the single reaction mixture. This method allows DNA to be amplified from any species or organism. It is understood that the present disclosure encompasses sequence independent amplification of DNA from any source, including but not limited to human, animal, plant, yeast, viral, eukaryotic, and prokaryotic DNA.
The present disclosure also offers an improved method for processing DNA samples on a solid medium. Other known methods of preparing DNA samples stored on a solid medium for genetic analysis or DNA amplification are inefficient and inconsistent, thereby limiting the usefulness of the information obtained from the DNA samples. The method of the present disclosure seeks to overcome the drawbacks inherent in these other methods by greatly simplifying the preparation of DNA samples and improving the DNA for subsequent analysis. The method of the present disclosure precipitates the DNA sample on a solid medium using methods well known to those of skill in the art. In a preferred embodiment, the DNA sample is a bloodstain on a solid medium. The DNA processed according to the presently disclosed method can be subsequently subjected to DNA amplification using the presently disclosed methods and/or genetic analysis. This disclosed precipitation method produces more consistent results, reduces the cost of high-throughput operations, and improves the quality of DNA amplified from the DNA sample.
The present disclosure includes methods of amplifying DNA from a DNA sample. In a preferred embodiment, the method of amplifying DNA uses a reaction mixture that contains a DNA sample; a first primer with a random sequence of nucleotides at its 3xe2x80x2 end and a generic sequence 5xe2x80x2 of the random nucleotides; and a second primer that has the generic sequence of the first primer but lacks the random sequence of the first primer. In a preferred embodiment, a single reaction mixture is used. An example of a first primer is the sequence designated SEQ ID NO:1, and an example of the second primer is the sequence designated SEQ ID NO:2. Preferably, the reaction mixture contains other components that are necessary for DNA amplification, which are well known to those of skill in the art.
The DNA sample in the reaction mixture is subjected to DNA amplification by a first DNA polymerase, wherein the first primer anneals to the DNA to allow the first DNA polymerase to synthesize a complementary DNA strand from the 3xe2x80x2 end of the first primer to produce a DNA product. The steps for DNA amplification by the first DNA polymerase are denaturing the DNA product; annealing the first primer with the DNA to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the first DNA polymerase to extend the primer and synthesize the DNA product. Preferably, these DNA amplification steps are repeated at least one time. In a preferred embodiment, the annealing temperature and the incubating temperature are the same. In another preferred embodiment, the DNA product produced by the DNA amplification is flanked by the generic sequence and reverse complement of the generic sequence.
This DNA product is then subjected to DNA amplification by a heat-stable DNA polymerase, wherein the second primer anneals to the DNA product and the heat-stable DNA polymerase synthesizes a complementary DNA strand from the 3xe2x80x2 end of the second primer to produce a second DNA product. The steps for DNA amplification by the heat-stable DNA polymerase are denaturing the DNA product; annealing the second primer with the DNA product to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the heat-stable DNA polymerase to synthesize the second DNA product. Preferably, these DNA amplification steps are repeated about 30 to about 35 times, and more preferably about 40 times. In a preferred embodiment, the second DNA product is flanked by the generic sequence and the reverse complement of the generic sequence. In another preferred embodiment, the annealing temperature is higher than the optimal annealing temperature of the random sequence of nucleotides at the 3xe2x80x2 end of the first primer.
In a preferred embodiment, the first DNA polymerase has 5xe2x80x2 to 3xe2x80x2 exonuclease activity or primer displacement activity. The first DNA polymerase is preferably E.coli DNA polymerase I, and the heat-stable DNA polymerase is preferably Taq DNA polymerase. In another preferred embodiment, the first DNA polymerase and the heat-stable DNA polymerase are the same DNA polymerase, and that DNA polymerase is Taq DNA polymerase.
In another preferred embodiment, the first primer has about 4 to about 8 random nucleotides at its 3xe2x80x2 end. In a more preferred embodiment, the first primer has about 6 random nucleotides at its 3xe2x80x2 end. Preferably the generic sequence of the first primer is about 15 to about 28 nucleotides in length, or more preferably about 20 to about 25 nucleotides in length. The random nucleotides of the first primer may also be G:C rich or A:T rich, to preferably amplify certain regions of the DNA sample. Finally, the generic sequence of the first primer preferably will have a single or multiple restriction enzyme recognition site, which will facilitate subcloning of the amplified DNA products.
In a preferred embodiment, the DNA sample is genomic DNA, microdissected chromosome DNA, yeast artificial chromosome (YAC) DNA, cosmid DNA, phage DNA, P1 derived artificial chromosome (PAC) DNA, or bacterial artificial chromosome (BAC) DNA. In another preferred embodiment, the DNA sample is mammalian DNA, plant DNA, yeast DNA, viral DNA, or prokaryotic DNA. In yet another preferred embodiment, the DNA sample is obtained from a human, bovine, porcine, ovine, equine, rodent, avian, fish, shrimp, plant, yeast, virus, or bacteria. Preferably the DNA sample is genomic DNA, wherein the method of amplifying DNA includes DNA amplification with a fluorescent label. In another preferred embodiment, the DNA sample is bovine DNA. Preferably, the DNA sample is tissue on a solid medium, wherein the tissue is blood, preferably in the form of a bloodstain. In a preferred embodiment, the solid medium is filter paper, wherein the filter paper is chemically treated, for example FTA(trademark) paper. In a preferred embodiment the bloodstain is from a mammal, and the mammal is preferably human, bovine, or porcine. The DNA sample may be obtained from many sources well known to those of skill in the art, including but not limited to a buccal swab, a nose swab, blood, cord blood, amniotic fluid, embryonic tissue, hair, endothelial cells, hoof clippings, or fingernail clipping.
In another preferred embodiment, the method of amplifying DNA further includes genotype analysis of the amplified DNA product. Alternatively, the method of amplifying DNA preferably further includes identifying a single nucleotide polymorphism (SNP) in the amplified DNA product. In preferred embodiments, a SNP may be identified in the DNA of an organism by a number of methods well known to those of skill in the art, including but not limited to identifying the SNP by DNA sequencing, by amplifying a PCR product and sequencing the PCR product, by Oligonucleotide Ligation Assay (OLA), by Doublecode OLA, by Single Base Extension Assay, by allele specific primer extension, or by mismatch hybridization. Preferably the identified SNP is associated with a phenotype, including disease phenotypes and desirable phenotypic traits. The amplified DNA generated by using the disclosed method of DNA amplification may also preferably be used to generate a DNA library, including but not limited to genomic DNA libraries, microdissected chromosome DNA libraries, BAC libraries, YAC libraries, PAC libraries, cDNA libraries, phage libraries, and cosmid libraries.
Another aspect of the present disclosure is a preferred method of amplifying DNA that uses a reaction mixture with a DNA sample; a first primer with a random sequence of nucleotides at its 3xe2x80x2 end and a generic sequence 5xe2x80x2 of the random nucleotides; a second primer with the generic sequence of the first primer and lacking the random sequence of the first primer; and a heat-stable DNA polymerase. In a preferred embodiment, a single reaction mixture is used. Preferably, the reaction mixture also contains other components that are necessary for DNA amplification, which are well known to those of skill in the art. In a preferred embodiment, the heat-stable DNA polymerase is Taq DNA polymerase, the DNA sample is a bloodstain on a solid medium, and the DNA sample is preferably dehydrated on the solid medium.
The DNA sample in the reaction mixture is subjected to DNA amplification wherein the first primer anneals to the DNA to allow the heat-stable DNA polymerase to synthesize a complementary DNA strand from the 3xe2x80x2 end of the first primer to produce a DNA product. The steps for DNA amplification by the heat-stable DNA polymerase are denaturing the DNA product; annealing the first primer with the DNA to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the heat-stable DNA polymerase to synthesize the DNA product. Preferably, these DNA amplification steps are repeated at least one time. In a preferred embodiment, the annealing temperature and the incubating temperature are the same. In another preferred embodiment, the DNA product produced by the DNA amplification is flanked by the generic sequence and reverse complement of the generic sequence.
This DNA product is then subjected to DNA amplification by the heat-stable DNA polymerase, wherein the second primer anneals to the DNA product and the heat-stable DNA polymerase synthesizes a complementary DNA strand from the 3xe2x80x2 end of the second primer to produce a second DNA product. The steps for DNA amplification by the heat-stable DNA polymerase are denaturing the DNA product; annealing the second primer with the DNA product to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the heat-stable DNA polymerase to synthesize the second DNA product. Preferably, these DNA amplification steps are repeated about 30 to about 35 times, and more preferably about 40 times. In a preferred embodiment, the second DNA product is flanked by the generic sequence and reverse complement of the generic sequence. In another preferred embodiment, the annealing temperature is higher than the optimal annealing temperature of the random sequence of the first primer.
In another preferred method of amplifying DNA, a reaction mixture is provided that has a DNA sample, wherein the DNA sample is a tissue on a solid medium; a first primer with a random sequence of nucleotides at its 3xe2x80x2 end and a generic sequence 5xe2x80x2 of the random nucleotides; a second primer with the generic sequence of the first primer and lacking the random sequence of the first primer; and a heat-stable DNA polymerase. In a preferred embodiment, a single reaction mixture is used. In a preferred embodiment, the tissue on the solid medium is blood. In another preferred embodiment the solid medium is filter paper, and the filter paper is chemically treated. The DNA sample is preferably dehydrated on the filter paper. Preferably, the reaction mixture also contains other components that are necessary for DNA amplification, which are well known to those of skill in the art. In a preferred embodiment, the heat-stable DNA polymerase is Taq DNA polymerase, the DNA sample is a bloodstain on a solid medium, and the DNA sample is preferably dehydrated on the solid medium.
The DNA sample in the reaction mixture is subjected to DNA amplification by a heat-stable DNA polymerase, wherein the first primer anneals to the DNA to allow the heat-stable DNA polymerase to synthesize a complementary DNA strand from the 3xe2x80x2 end of the first primer to produce a DNA product. The steps for DNA amplification by the heat-stable DNA polymerase are denaturing the DNA product; annealing the first primer with the DNA to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the heat-stable DNA polymerase to synthesize the DNA product. Preferably, these DNA amplification steps are repeated at least one time. In a preferred embodiment, the annealing temperature and the incubating temperature are the same. In another preferred embodiment, the DNA product produced by the DNA amplification is flanked by the generic sequence and reverse complement of the generic sequence.
This DNA product is then subjected to DNA amplification by the heat-stable DNA polymerase, wherein the second primer anneals to the DNA product and the heat-stable DNA polymerase synthesizes a complementary DNA strand from the 3xe2x80x2 end of the second primer to produce a second DNA product. The steps for DNA amplification by the heat-stable DNA polymerase are denaturing the DNA product; annealing the second primer with the DNA product to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the heat-stable DNA polymerase to synthesize the second DNA product. Preferably, these DNA amplification steps are repeated about 30 to about 35 times, and more preferably about 40 times. In a preferred embodiment, the second DNA product is flanked by the generic sequence and reverse complement of the generic sequence. In another preferred embodiment, the annealing temperature is higher than the optimal annealing temperature of the random sequence of nucleotides at the 3xe2x80x2 end of the first primer.
Another aspect of the present disclosure is a preferred method of identifying a polymorphism, which uses a reaction mixture with a DNA sample; a first primer with a random sequence of nucleotides at its 3xe2x80x2 end and a generic sequence 5xe2x80x2 of the random nucleotides; and a second primer with the generic sequence of the first primer and lacking the random sequence of the first primer. In a preferred embodiment, a single reaction mixture is used. Preferably, the reaction mixture also contains other components that are necessary for DNA amplification, which are well known to those of skill in the art. In a preferred embodiment, the amplified DNA products are analyzed to identify a polymorphism, and preferably the polymorphism is a single nucleotide polymorphism (SNP). The methods of identifying SNPs are well known to those of skill in the art. In a preferred embodiment of identifying a SNP, the SNP is identified by DNA sequencing, Oligonucleotide Ligation Assay (OLA), Doublecode OLA, Single Base Extension Assay, allele specific primer extension, or mismatch hybridization.
The DNA sample in the reaction mixture is subjected to DNA amplification by a first DNA polymerase, wherein the first primer anneals to the DNA to allow the first DNA polymerase to synthesize a complementary DNA strand from the 3xe2x80x2 end of the first primer to produce a DNA product. The steps for DNA amplification by the first DNA polymerase are denaturing the DNA product; annealing the first primer with the DNA to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the first DNA polymerase to synthesize the DNA product. Preferably, these DNA amplification steps are repeated at least one time. In a preferred embodiment, the DNA product produced by the DNA amplification is flanked by the generic sequence and reverse complement of the generic sequence.
This DNA product is then subjected to DNA amplification by a heat-stable DNA polymerase, wherein the second primer anneals to the DNA product to allow the heat-stable DNA polymerase to produce amplified DNA products. The amplified DNA products are produced when the heat-stable DNA polymerase synthesizes a complementary DNA strand from the 3xe2x80x2 end of the second primer annealed to the DNA product. The steps for DNA amplification by the heat-stable DNA polymerase are denaturing the DNA product; annealing the second primer with the DNA product to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the heat-stable DNA polymerase to synthesize the amplified DNA products. The amplified DNA products are then preferably analyzed to identify a polymorphism. Preferably, these DNA amplification steps are repeated about 30 to about 35 times, and more preferably about 40 times. In a preferred embodiment, the amplified DNA products are flanked by the generic sequence and reverse complement of the generic sequence. In another preferred embodiment, the annealing temperature is higher than the optimal annealing temperature of the random sequence of nucleotides at the 3xe2x80x2 end of the first primer.
Another aspect of the present disclosure is a preferred method of amplifying DNA that uses a reaction mixture with a DNA sample to be amplified; a first primer with a random sequence of nucleotides at its 3xe2x80x2 end and a generic sequence 5xe2x80x2 of the random nucleotides; and a second primer with the generic sequence of the first primer and lacking the random sequence of the first primer. Preferably, the reaction mixture also contains other components that are necessary for DNA amplification, which are well known to those of skill in the art. In a preferred embodiment, the DNA sample is tissue on a solid medium, and the DNA sample is preferably dehydrated on the solid medium.
The DNA sample in the reaction mixture is heated to a temperature that denatures the DNA to be amplified, cooled to a temperature that allows the random sequence of the first primer to hybridize to its complement DNA, and incubated to allow synthesis of a DNA product by a DNA polymerase. In a preferred embodiment the DNA polymerase is Taq DNA polymerase. Preferably, the steps of heating, cooling, and incubating the reaction mixture are repeated at least one time. In another preferred embodiment, the DNA product is flanked by the generic sequence and reverse complement of the generic sequence.
A series of DNA amplification reactions are performed with the DNA product, wherein the annealing step is at a temperature that selects for the generic sequence of the second primer hybridizing to complement DNA in the DNA product over the random sequence of the first primer hybridizing to complement DNA in the DNA product. Preferably, the DNA amplification reactions involve a heat-stable DNA polymerase synthesizing a complementary DNA strand from the 3xe2x80x2 end of the second primer to produce amplified DNA products. The steps for DNA amplification by the heat-stable DNA polymerase are denaturing the DNA product; annealing the second primer with the DNA product to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the heat-stable DNA polymerase to synthesize amplified DNA products. Preferably, the series of DNA amplification reactions includes about 30 to about 35 reactions, and more preferably about 40 reactions. In a preferred embodiment, the products of the DNA amplification reactions are flanked by the generic sequence and reverse complement of the generic sequence.
A preferred embodiment of the present disclosure is a method of amplifying a DNA sample on a solid medium that involves precipitating the DNA sample on the solid medium and subjecting the precipitated DNA to DNA amplification to produce amplified DNA products. Preferably the solid medium is filter paper, and the filter paper is chemically treated. In a preferred embodiment, the DNA sample is dehydrated on the filter paper. In another preferred embodiment, the DNA sample is tissue, and the tissue is blood.
The DNA sample on a solid medium is preferably precipitated with salt and alcohol, and rinsed with alcohol. Preferably, the salt used to precipitated the DNA is sodium acetate, potassium acetate, ammonium acetate, sodium chloride, or potassium chloride; preferably the alcohol used is ethanol or isopropanol. In preferred embodiments, the produced amplified DNA products are subjected to genotype analysis, or a polymorphism is identified in the DNA products, preferably a single nucleotide polymorphism (SNP). The SNP may be identified by a number of techniques well known to those of skill in the art, including preferably DNA sequencing, Oligonucleotide Ligation Assay (OLA), Doublecode OLA, Single Base Extension Assay, allele specific primer extension, or mismatch hybridization.
Another aspect of the present disclosure is a preferred method of identifying a polymorphism, which precipitates a DNA sample on a solid medium, and uses a reaction mixture with the precipitated DNA sample; a first primer with a random sequence of nucleotides at its 3xe2x80x2 end and a generic sequence 5xe2x80x2 of the random nucleotides; and a second primer with the generic sequence of the first primer and lacking the random sequence of the first primer. In a preferred embodiment, a single reaction mixture is used. Preferably, the reaction mixture also contains other components that are necessary for DNA amplification, which are well known to those of skill in the art. In a preferred embodiment, the amplified DNA products are analyzed to identify a polymorphism, and preferably the polymorphism is a single nucleotide polymorphism (SNP). The methods of identifying SNPs are well known to those of skill in the art. In a preferred embodiment of identifying a SNP, the SNP is identified by DNA sequencing, Oligonucleotide Ligation Assay (OLA), Doublecode OLA, Single Base Extension Assay, allele specific primer extension, or mismatch hybridization.
The DNA sample in the reaction mixture is subjected to DNA amplification by a first DNA polymerase, wherein the first primer anneals to the DNA to allow the first DNA polymerase to synthesize a complementary DNA strand from the 3xe2x80x2 end of the first primer to produce a DNA product. The steps for DNA amplification by the first DNA polymerase are denaturing the DNA product; annealing the first primer with the DNA to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the first DNA polymerase to synthesize the DNA product. Preferably, these DNA amplification steps are repeated at least one time. In a preferred embodiment, the DNA product produced by the DNA amplification is flanked by the generic sequence and reverse complement of the generic sequence.
This DNA product is then subjected to DNA amplification by a heat-stable DNA polymerase, wherein the second primer anneals to the DNA product to allow the heat-stable DNA polymerase to produce a second DNA product. The second DNA product is produced when the heat-stable DNA polymerase synthesizes a complementary DNA strand from the 3xe2x80x2 end of the second primer annealed to the DNA product. The steps for DNA amplification by the heat-stable DNA polymerase are denaturing the DNA product; annealing the second primer with the DNA product to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the heat-stable DNA polymerase to synthesize the second DNA product. The amplified DNA product is then preferably analyzed to identify a polymorphism. Preferably, these DNA amplification steps are repeated about 30 to about 35 times, and more preferably about 40 times. In a preferred embodiment, the second DNA product is flanked by the generic sequence and reverse complement of the generic sequence. In another preferred embodiment, the annealing temperature is higher than the optimal annealing temperature of the random sequence of nucleotides at the 3xe2x80x2 end of the first primer.
In a preferred embodiment, the solid medium is filter paper, and the filter paper is chemically treated. In another preferred embodiment, the DNA sample is dehydrated on the filter paper. Preferably, the DNA sample is tissue, and the tissue is blood. The DNA sample on a solid medium is preferably precipitated with salt and alcohol, and rinsed with alcohol. Preferably, the salt used to precipitated the DNA is sodium acetate, potassium acetate, ammonium acetate, sodium chloride, or potassium chloride; preferably the alcohol used is ethanol or isopropanol.
The DNA amplification methods of the present disclosure will be useful for amplifying small amounts of DNA, which will allow multiple sites in the DNA sample to be genotyped for high-throughput screening. Additionally, the present method will allow for the rapid construction of band specific painting probes for any chromosomal region, and can also be used to microdissect and amplify unidentifiable chromosomal regions or marker chromosomes in abnormal karyotypes. The presently disclosed method will also allow for the rapid cloning of amplified DNA for sequencing or generating DNA libraries. Thus, the method will not only be a valuable tool for genotype analysis and high-throughput screening, it should also be a valuable tool in cytogenetic diagnosis.